HEPATOZOON KISRAE N. SP. INFECTING THE LIZARD AGAMA STELUO

IS TRANSMITTED BY THE TICK HYALOMMA CF. AEGYPTIUM

PAPERNA E*. KREMER-MECABELL T.* & FINKELMAN S.*

Summary :

Hepatozoon kisrae n. sp. was found infecting a starred lizard at a site in southeastern Samaria, Palestine. These lizards were also hosts to the ixodid tick Hyolomma cf. aegyptium, which was demonstrated to be the vector of this hemogregarine. Hepatozoon and tick infections occurred in lizards within a very restricted locality; at a second site, nearby, ticks occurred without Hepatozoon infection. Micro- and macromeronts occurred mainly in the lungs, while cyst-like merogonic stages, mainly dizoic, occurred in the liver. Mature intraerythrocytic gametocytes were stout and encapsulated. Development from oocysts to sporocysts took place in the tick hemocoel, and was examined by transmission electron microscopy. Lizards were successfully infected when fed on sporocyst-infected ticks or viscera of infected lizards. Ticks become infected when fed on infected lizards; sporogony was complete when the ticks reached adult stage, over 4 0 days after initial attachment.

KEY WORDS : Hepatozoon kisrae n. sp., Agama stellio, Hyalomma cf. aegyptium, development, transmission, sporogony, ultrastructure.

Résumé : HEPATOZOON KISRAE N. SP., PARASITE DU LÉZARD AGAMA STELLIO EST TRANSMIS PAR LA TIQUE HYALOMMA CF. AEGYPTIUM

Hepatozoon kisrae n. sp. a été découvert chez Agama stellio dans une localité du sud-est de la Samarie (Palestine). Ces Lézards sont égalemant les hôtes de Hyalomma cf. aegyptium. Il est démontré que cet Ixodidé est le vecteur de l'Hémogrégarine. Le cycle ne s'effectue que dans une zone précise : les Tiques d'une région voisine sont indemnes. Les micro et macromérontes se trouvent essentiellement dans les poumons, alors que les kystes (la plupart contenant deux cystozoïtes) siègent dans le foie. Les gamétocytes intraérythrocytaires mûrs, de forme trapue, sont encapsulés. Le développement depuis l'oocyste jusqu'au sporocyste, qui a lieu dans l'hémocéle de la Tique, a été étudié en microscopìe électronique. Les Lézards s'infectent par ingestion, soit de Tiques infectées de sporocystes, soit de viscères de Lézards infectés. Les Tiques s'infectent par repas sur Lézards infectés ; la sporogonie est achevée 40 jours après la fixation de la nymphe, lorque le stade adulte est atteint.

MOTS CLES : Hepatozoon kisrae n. sp., Agama stellio, Hyalomma cf. aegyptium, développement, transmission, sporogonie, ultrastructure.

INTRODUCTION

Ticks and mites have been shown to vector Hepa­

tozoon o f mammalian (Miller, 1908; Hoogstraal,

1961; Furman, 1966; Mathew et al, 1999) and

avian hosts (Bennet et al, 1992); there is, however,

only one report of reptile-host Hepatozoon sporogonic

development in a tick (in Amblyoma dissimilae, Ball

et al., 1969) . Hemogregarines o f the genus Hemolivia,

infecting toads (Bufo marinus), lizards (Tiliqua rugosa)

and land tortoises (Testudo graecd) are tick-transmitted,

and differ considerably in their course of development

in their vector from species o f Hepatozoon infecting

reptiles transmitted via insects, notably mosquitoes. In

the first, in the tick host, oocysts remain in the gut cells

to yield a progeny o f mobile sporokinetes, which re­

enter the gut tissue to proceed sporogenesis (Petit et

al., 1990; Smallridge & Paperna, 1997; Landau & Paperna,

* Department of Animal Sciences, Faculty of Agricultural, food and environmental sciences, The Hebrew University of Jerusalem, Rehovot 76-100 Israel. Correspondence: Paperna I. Tel.: 972 8 9489945 - Fax: 972 8 9465763. E-mail: paperna@agri.huji.ac.il

1997; Smallridge & Paperna, 2000 a,b). In the latter,

developing in dipteran insects (predominantly mos­

quitoes), oocysts escape into the hemocoel , sporocysts

develop and remain to sporulate within the oocyst

body (Lowichick et al, 1993)- Hepatozoon o f mam­

malian and avian hosts, unergo similar development

into sporocyst loaded oocysts in their vector's hemo­

coel (Bennet et al, 1992; Mathew et al., 1999). A few

species o f named Hepatozoon species have been

reported to be transmitted via mites (Lewis & Wagner,

1964; Allison & Desser, 1981), their potential affiliation,

however, with the mite-transmitted hemogregarinid

genus Karyolysus (Reichenow, 1921; Svahn, 1975) needs

to be re-examined.

The starred lizard, Agama stellio, is widespread throu­

ghout the Middle East (Haas, 1951) . Desser & Yeku-

tiel (1986-1987) examined blood smears from starred

lizards collected from different localities in Israel and

Palestine and found two morphologically distinct types

o f undescribed hemogregarines, stout and elongate.

The elongate hemogregarine is an as yet undescribed

specie o f Hepatozoon (vide sp. A), which was trans­

mitted experimentally via mosquitoes (Culexpipiens,

Finkelman & Paperna, unpublished).

Memoir 17 Parasite, 2002, 9, 17-27

Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/200209117

PAPERNA I., KREMER-MECABELL T. & FINKELMAN S.

In this communication we describe the species forming the stout gametocytes, Hepatozoon kisraen. sp., which infects the starred lizard at sites in southeastern Samaria, Palestine. These lizards are also the hosts to an ixodid tick Hyalomma cf. aegyptium, which is demonstrated to vector this hemogregarine.

MATERIALS AND METHODS

Starred lizards (A. stellio) (n = 27) were captured bet­ween September 23, 1999 to August 24, 2000 on the southeastern slopes o f the Samarian mountains

in Palestine (1978 edition map of Israel grids: 155-170 N/182-187E), at altitudes of 550 to 700 m above sea level, in a semiarid Mediterranean habitat (~ 350 mm annual rainfall) consisting o f rocky areas and terrace-fenced olive groves. The collection sites were the fol­lowing: a) a stone-fenced olive grove, < 1,000 m 2 in area ("Kisra", grid 166N/184E); b ) its adjoining grounds, at a perimeter o f 500 m ("near Kisra"); c ) at roadsides along two km on Akraba-Kisra road; d) a site situated 13 km down this road to the south ("Roman camp") and e ) two localities situated three and five km on the road branching eastwards from Kisra (sites c-e are listed as "elsewhere" in table I). Blood smears were obtained by clipping the lizard's toe tip. Ticks were opened by incision and their vis­ceral contents were examined by direct light micro­scopy and from stained smears. Prepared blood films and smears were air-dried, fixed in absolute methyl alcohol and stained for one hour in Giemsa (10 % in phospha te buffer pH 7 .4 ) . Levels o f parasitaemia defined as: ± light (< 0.5 % ) , + low (0.5-3 % ) . ++ mode­rate (3-9 % ) , +++ high (> 9 % ) were determined from counted 50 fields at x 1,000 magnification (~ 400 ery­throcytes per field). From moribund, euthanized and freshly dead lizards, tissue samples of the liver, lungs and sometimes spleen were fixed for histological examination in 10 % neu­tral-buffered formalin. After dehydration in graded ethanols, the tissues were embedded in glycol-metha-crylate medium (GMA of Agar Comp., UK). Sections, 3 pm thick, were cut with a glass knife on a Sorval J B 4 microtome and stained with either Meyer's hae-

malum-eosin or, after a 20 min incubation in Bouin's solution and a rinse with water and 70 % ethanol, with 10 % Giemsa, in phosphate buffer pH 7.4, for one hour. Captive lizards were kept, each in a glass terrarium 50 x 30 x 30 cm in size under continuous illumination at room temperature of 25-31° C and were fed on meal­worms supplemented by chopped chicken liver and meat. Experimental infection of lizards was carried out 1) through their feeding on sporocysts dissected from naturally and laboratory infected ticks (H. cf. aegyp­tium); 2) by feeding on blood clots and viscera (mainly liver) o f euthanized infected lizards. Together with the starred lizards, two geckoes (Ptyodactylus basselquistii) were also fed on viscera of infected ticks. Infection-free lizards were obtained from habitats other than the enzootic location, and checked over two week period to verify the absence of infection. Visceral contents of the ticks: males, semi-engorged adult stages and nymphs released by small incision, were exa­mined directly and/or from air-dried, methanol-fixed, Giemsa- stained smears. Positive ticks were used either for an ultrastructural study, or to infect lizards via fee­ding.

Engorged female ticks removed from captured lizards were kept in individual vials for oviposition, in slightly moistened chambers (~ 70-90 % RH) at ambient room temperature of 25-31° C. Laid eggs were incubated, and engorged larvae and nymphs were allowed to molt in vials under same conditions o f temperature and humi­dity. Hungry larvae, nymphs and adults were stored in a 16° C incubator. Lizards, left free or caged in a net-box, were exposed to larvae or nymphs in contai­ners with the rims of their tops aligned by sticky bands. For transmission electron microscopic (TEM) exami­nation, ticks were immersed in 2.5 % glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4), tick abdomen was then sectioned at the anterior and posterior extremi­ties, and either left that way or followed by progres­sive separation of the digestive tract from the chitinous envelope o f the tick. The tick material left in the same fixative for 24 h at 4° C, was then rinsed in 0.1 M caco­dylate buffer and postfixed in 1 % osmium tetroxide in the same buffer for one hour. After rinsing in the

Kisra Near Kisra Elsewhere Localities (site a) (site b) (sites c-e)

Total no lizards examined 10 2 15 No. lizards infested with ticks 7 1 4 No. infected with H. kisrae 9 0 0 No. infected with Hepatozoon sp. A 0 0 3 No. lizards with Hepatozoon-infected ticks 6 0 0

Table I. - Summary of numbers of starred lizards examined from localities in Samaria, found infected by Hepatozoon spp. and found infested by Hepatozoon-infected and non-infected ticks.

Mémoire 18- Parasite, 2002, 9, 17-27

TICK TRANSMITTED HEPATOZOON OF AGAMA

buffer, the material was dehydrated in graded ethanols and embedded in Agar 100 medium (Agar Scientific, Ltd., UK). Thin sections, cut on a Reichert Ultracut microtome with a diamond knife were stained on grids with uranyl acetate and lead citrate, and exa­mined with a J eo l 100CX TEM. Semithin sections cut on the same microtome were stained with toluidine blue for examination by light microscopy.

RESULTS

SPATIAL D I S T R I B U T I O N O F HEPATOZOON A N D T I C K S

A M O N G L I Z A R D S

All but one o f the lizards (n = 10) caught in the olive grove in Kisra were infected with Hepato­zoon kisraen. sp.; of these, seven were infested

with the tick Hyaloma cf. aegyptium (Table I ) . The only lizard found non-infected by H. kisrae was free from ticks. Ticks removed from the H. kisrae-infected lizards hosted either mature sporocysts or premature sporogony stages (Table I I ) , with the exception of ticks removed from the low-infected lizard (No. 2, Table I I ) , which were not infected. None o f the lizards caught outside the olive grove, either nearby (n = 2) , or else­where (n = 15) were infected with H. kisrae. Three o f the latter lizards were infected by the mosquito-trans­mitted Hepatozoon sp. A. Ticks were found on one lizard from site b (a larva), on two in site c (a nymph and a male, both lizards were also infected with Hepa­tozoon sp. A), and on two lizards in site d (a male and two unengorged females); none were found hosting developing stages o f Hepatozoon (Table I I ) .

DESCRIPTION O F HEPATOZOON KISRAE N . SP.

Hosts: Starred lizard, Agama stellio; the tick Hyalomma cf. aegyptium.

Type locality: Olive grove at Kisra (grids 182 E / 1 6 2 N), southeast Samaria, Palestine. Etymology: Named after the type-host locality.

Stages in the starred lizard M e r o g o n i c s tages dividing by p o l y e n d o d y o g e n y (Fig. 1A), seen in the lungs, were encapsulated (29-39 x 15-22 pm in size, n = 7 ) inside endothelial cells, com­prised o f either macromeronts dividing into 4 to 16, 11-16 x 3-4 um merozoites (Fig. 1 A-D), or microme-ronts dividing to 16 to 32, 8-11 x 1.5-2 pm merozoites (Fig. 1, A, E-G). In the liver occurred the cyst-type meronts or cysto-zoites, with two ("dizoic", 19-24 x 12-17 pm in size, n = 7, Fig. 1H-L), four (20-28 x 10-15 pm in size, n - 7, Fig. 2A) and exceptionally eight zoites. These cysts were lodged within melanomacrophage centers (MMC), evidently inside macrophages. The zoite (12-15 x 2-5 um in size, n = 12) couples were apparently formed by endodyogeny. They were enclosed in a meront resi­duum (the cystic body) filled with vacuolar matrix (visible only in histology). Some dizoites contained a blue-staining inclusion, which might be a crystalline body (Fig. I I , L). Exceptionally large zoites, 22.2 x 3.8 pm, showed tapering, deep staining, anterior end (not shown).

The liver also contained a few merogonic stages, mero­gonic (polyendodyogenic) division inside an endo­thelial cell is shown in Figure 2B; macro and micro-merogonic stages (both 23-29 x 15-18 pm in size) contained up to 16 (Fig. 2C) and 32 zoites, respecti­vely. Liver tissue also contained free, single, un-divided meronts (Fig. 2D), reaching a size o f 16-17 x 4-7 pm (n = 2) . Four lizards, two naturally infected, one infected by ingestion of a tick and one by infected lizard viscera (1 ) , kept for 37 to 167 days, when necropsied, all demonstrated the presence o f cyst-type meronts in the liver. In the two lizards also examined for lung infec-

Infected by No. No. No. With mature Lizard marking Date H. kisrae ticks present examined infected sporoôcysts

1 September 23. 1999 + 0 2 September 23, 1999 i 1M. 2seN 1M, IseN 0

3 September 23, 1999 ++ 1M, IF, 2seN, leL 1M, IF 2 2 (.11 October 15, 1999 0 0 8C May 5, 2000 +++ 3M, IF, 3eL 1M, IF 2 2 9C May 5, 2000 +++ 3M, 4F 1M, 3F 1M, 2F 1M

10C May 5, 2000 ^ + 0 11C May 24, 2000 1M, IF 1M, IF 1M 1

2K August 24, 2000 lOseN 3seN, 1#M 3eN, 1#M 1#M 3K August 24, 2000 + 3seN, 1M 1M, 1#M, 1#F 1M, 1#M, 1#F 1M, 1#M, 1#F

Levels of parasitaemia: ± light (< 0.5 % ) , + low (0.5-3 % ) , ++ moderate (3-9 % ) , +++ high (> 9 % ) .

Table 11. - Analysis of the state of infection of starred lizards and ticks collected in Kisra olive grove (site a); ticks: M-male, F-female, N-nymph, L-larva, e-engorged, se-semiengorged. * Immature ticks were allowed to molt before examination.

Parasite, 2002, 9, 17-27 Mémoire 19

PAPERNA I.. KREMER-MECABELL T. & FINKELMAN S.

Fig.1. - Merogonic stages of Hepatozoon kisrae n.sp. from lungs and liver of Agama stellio (x 1,300). A-G, stages in lungs: A. macrome-ronts in division (a) and divided micromeronts (i); B. Macromeronts after division; C. Macromeronts prior division; D. Post-division macro-meront formation; E. Micromeronts prior division; F,G. Post division micromeront formations. H-L: Dizoic cyst-type meronts in the liver (L, merozoites show blue-staining crystalline-like bodies, marked with arrows).

20 Mémoire Parasite, 2002, 9, 17-27

Fig. 2. - Stages of Hepatozoon kisrae n. sp. from Agama stellio (x 1,300). A-D, from the liver: A. Four-zoite cyst-like meront; B. Meront dividing by polyendodyogeny; C. Polyzoic meront (macromeront); D. Undivided meront (arrow) and intraerythrocytic gametocytes (d). E-G: Gametocytes in the blood. E. Oval young (a) and premature with spirale nucleus (c); F. Premature with dense nucleus (b) and mature, encapsulated (d); G. Mature encapsulated; H . Hepatozoon sp. A. in blood of A. stellio from site e.

Parasite, 2002, 9, 17-27 Mémoire 21

TlCK TRANSMITTED HEPATOZOON OF AGAMA

PAPERNA L, KREMIER-MECABELL T. & FINKELMAN S.

Lizard marking Date infected Onset of

parasitaemia Necropsy Liver Lungs Blood

1 natural Collected September 23, 1999

(before 23.9) after 167 days z2 + Mi & Ma ++ Gam ++

3 natural Collected September 23, 1999

(before 23.9) after 84 days z2 ++, z4,8 Mi & Ma ++++ Gam ++

2B November 7, 1999 by sporocysts

32 dpi 120 dpi z2 +++, z4, 8, Mi & Ma 16,32 +

? Gam ++

7B March 7, 1999 by lizard viscera

< 37 dpi 37 dpi z2+, z8 ± 7 Gam ++

Table III. - Protocol of necropsies of 4 infected starred lizards: Exoerythrocytic z-zoites (cystozoites), Mi-micromeronts, Ma-macromeronts, Gam-gametocytes in blood. Parasite stages in the tissue: ± scarce, + a few, ++ prevalent, +++ numerous, ++++ veiy numerous.

tion, macro- and micromeront infection coincided with the liver infection (Table III). Gametocytes at several stages of differentiation occur­red in the erythrocytes. The earliest ones were oblong, 6-8 x 4-5 pm (n = 4 ) , and subsequently oval, 7-9 x 5-6 pm, with a large central nucleus (Fig. 2E). Interme­diate stages (7-11 x 5-7 pm, n = 4 ) had a nucleus with spirally scattered chromatin (Fig. 2E, F) . The presu­mably fully mature stages were stout, banana-shaped, 12-15 x 2-5.5 pm in size (n = 9 ) , with a centrally located 4 to 5.5 um wide multilobed or homoge­neously packed nucleus; the cytoplasm was either stained homogeneously pale, or had a variable quan­tity o f vacuoles and granules (Fig. 2F, G ) . Fully mature gametocytes were encased within a hard capsule. The gametocytes were readily differentiated from the sym-patric species of Hepatozoon found in A. stellio b lood (Hepatozoon sp. A, Finkelman & Paperna, unpubli­shed), the latter being elongate, with inward-bent ends (Fig. 2H).

Development in the tick Syzygy was not detected. Oocysts, 67-74 x 47-54 pm (n = 4 ) in size, with expanded vacuolated cytoplasm, and a nucleus with a conspicuously large nucleolus, were found in the hemocoel , on the gut surface of engorged nymphs (Fig. 3A, B) . Via segmentation (Fig. 3 0 , sporoblasts formed aggregates o f sporocysts, 43-50 x 24-27 pm (n = 10) in size (Fig. 3D) . Early differentiated sporocysts in engorged nymphs showed the already split crystalline bodies (Fig. 3E) . Oocys t progeny consisted o f over 100 sporocysts. The formed sporo­cysts remained aggregated within the oocyst, forming a ball-like structures (200-230 x 230 pm in size) in the hemocoel (Fig. 3F) visible to the naked eye as white balls. Mature oocysts with ripe sporocysts were found in unengorged adult male and female ticks and in nymphs, which failed to complete engorgement. Indi­vidual sporocysts, encased in a hardened wall, varied in size from 34 x 24 to 6l x 23 pm, and contained 16 to 35 sporozoites (Fig. 3G) .

ULTRASTRIICTURAL OBSERVATIONS ON SPOROCYST DEVELOPMENT

Young sporocysts which had split from the sporoblast (Fig. 4A) gradually exhausted their stored amylopectin granules as well as their lipid vacuoles (Fig. 4 B ) . Small granular aggregates - the anlagen of crystalloid bodies, enclosed within rough endoplasmic reticulum (ER), grew to large inclusions o f granular matrices (Fig. 4 B , C), which became consolidated into arrays o f crystal­loids (Fig. 4D, E) . The ER network also incorporated numerous tubular mitochondria (Fig. 4 B ) . Small elec­tron-dense bodies remained in the sporocyst cyto­plasm to late stage of differentiation (Fig. 4A, B , D) . The rough ER, which accompanied the forming crys­talloid bodies, disappeared when the crystalloid arrays became consolidated (Fig. 4D, E) . The single nucleus of the formed sporocysts divided. All formed nuclei had a conspicuous, often aggregated nucleolus (Fig. 4D, E) . At this stage encapsulation started, the future hard wall consolidated above the plasmalemma, and became superimposed by a fine veil (Fig. 4F) . Increased har­dening o f the sporocyst wall accelerated its resistence to fixation and impregnation, which eventually pre­cluded further ultrastructural analysis.

EXPERIMENTAL INFECTIONS

From ticks to lizards Six starred lizards (all adults), from sites other than Kisra, verified to be non-infected, were fed on sporo-cysts-infected H. cf. aegyptium ticks (Table IV). Five were fed, each on a tick removed from naturally infected lizard and one (7K) on a tick experimentally engorged on an infected lizard. The latter lizard deve­loped the same infection schedule as the ones fed on naturally infected ticks. All but one developed parasi­temia not later than 32 to 40 days post- infection (p.i), in the latter (1C), light infection with young gameto­cytes was detected only 90 days p.i. In two lizards blood already contained mature gametocytes by day

22 Mémoire Parasite, 2002, 9, 17-27

TICK TRANSMITTED HETATOZOON OF AGAMA

Fig. 3. - Developmental stages of Hepatozoon kisrae n. sp. in the tick Hyalomma cf. aegyptium. A-E, toluidine-stained semithin sections; F,G, unfixed, live: A,B. Oocysts, x 1,156 and x 462; C. Oocyst (o) dividing into sporocysts (s) x 500; D. Sporocysts x 887; E. Sporocysts with divided crystalline bodies x 939; F. Oocyst ball filled with ripe sporocysts x 324; G. Single sporocysts filled with sporozoites x 1,968.

Parasite, 2002, 9, 17-27 23 Mémoire

Fig. 4. - Electron microscopic images of sporocysts from infected ticks. A. Newly formed sporocyst filled with amylopectin granules, sho­wing anlagen of crystalloid bodies enclosed in rough ER (pr) mitochondria (m) and electron-dense bodies (d); x 7,150. B. Sporocyst with ER-aligned inclusion of pre-crystalloid granular bodies (r), showing also mitochondria (m) and lipid vacuoles (L); x 6,400. C. Sector of divided sporocysts showing a nucleus (n) with scattered nucleolus (ns), and ER (er) aligning the pre-crystalloid granular body (r): x 10,344. D,E. Sporocysts with several nuclei (n), arrayed crystalloid bodies (r), amylopectin granules (a) and electron-dense bodies (d); x 6000 and X 6, 460. F. Walling process of a sporocyst, the forming wall is invested by a veil (arrowhead); In addition to the formed crystalloid body (r) a crystalloid anlage (pr) enclosed in ER still occurs; a, amylopectin granules; x 15,300.

PAPERNA I., KREMER-MECABELL T. & FINKELMAN S.

24 Mémoire Parasite, 2002, 9, 17-27

TICK TRANSMITTED HEPATOZOON OF AGAMA

Date fed Dpi to first Dpi to mature Lizard marking Origin Date on sporocysts seen infection gametocytes

2B site d October 15, 1999 November 7, 1999 32 60 IC site c May 5, 2000 May 15, 2000 93 ~ IK site b August 24, 2000 October 17, 2000 40 4K site b August 24, 2000 October 26, 2000 40 5K site e August 24, 2000 September 3, 2000 37 7K site d August 24, 2000 September 3. 2000 < 37 37 (died)

37 p.i., in further two by day 40 p.i. and in one by day 60 p.i. The two geckoes fed simultaneously with I K and 4K starred lizards on sporocyst-infected tick viscera, failed to develop infection by day 40 p.i.

From starred lizard to starred lizard Three lizards (two juveniles and one adult from out­side Samaria) were fed on blood and liver obtained from a euthanized infected lizard (lizard 1, Table II) . Parasitaemia, including o f mature encapsulated game­tocytes was detected by day 37 in the adult and 47 days p.i. in both juvenile lizards.

From lizards to tick T w o H. kisrae- infected lizards, and three non infected ones were exposed to H. cf. aegyptium larvae, engor­ged larvae were obtained after 11-13 days post-expo­sure, some o f the larvae proceeded without descen­ding to the nymph stage and dropped off as engorged nymphs 30 to 38 days later. None of these, engorged larvae or hungry nymphs, recovered up to 40 days post-attachment were found infected. Infection was, however , found in spontaneously detached semi-engorged nymphs (n = 6) who failed to molt, exa­mined 41 days post initial attachment. Infection was already comprised of mature sporocysts with sporo-zoites. Adult ticks developing from molting engorged nymphs w e r e found infec ted by non-sporu la ted oocysts, as well as oocysts containing non-differentiated and mature sporocysts. These ticks were 41 to 77 days after initial exposure. All 10 dissected adult ticks and three engorged nymphs recovered after a feeding schedule on three Hepato­zoon sp. A.-infected lizards examined either 42 to 77 days (adults) or 20 to 42 days (engorged nymphs) after initial attachment were negative. One engorged nymph examined 22 days post-attachment contained Hepato­zoon sp. A. gametocytes.

Transovarian transmission Infection was not traced in any o f the progeny grown from ovigerous female removed from infected lizard from Kisra. Examined ticks were nymphs (3 ) and adults ( 1 0 ) engorged after feeding on three non-infected lizards.

DISCUSSION

A gama stellio, the starred lizard is abundant and widely distributed in the Near East and has become a peridomestic inhabitant (Haas, 1951).

On the otherhand the spatial distribution o f both ticks and H. kisrae infection in starred lizards appears to be very patchy, the present locality was restricted to a stone fence-enclosed area of less than 1,000 m 2 . Desser & Yekutiel (1986-1987) reported Hepatozoon infection by stout gametocytes , apparently conspecif ic with H. kisrae, in starred lizards from three localities: two in the same arid geographical subregion (in south-east Samaria), and one in the forested Mediterranean zone in west Jerusalem, with ~ 800 mm annual rainfall. Hya-lomma cf. aegyptium were never been found on the previously investigated starred lizards (Ostrovska & Paperna, 1987; Bristowetzki & Paperna, 1990) . As was found in our study, not all patches of tick infection necessarily generate active H. kisrae transmission. The species of Hepatozoon co-habiting starred lizards are readily distinguishable, and appears to demonstrate a strict specificity to their respective vector hosts, H. kis­rae, with the stout gametocytes, to H. cf. aegyptium and Hepatozoon sp. A, with the elongate gametocytes, to mosquitoes (Culex pipiens, Finkelman & Paperna, unpublished). Transmission by mosquitoes could favor a more continuous pattern o f distribution for Hepato­zoon sp. A. The spatial distribution o f this apparently more abundant species , however , is also patchy, though over seemingly a wider range o f habitats (Fin­kelman & Paperna, unpublished). H. kisrae appears to have narrow host specificity, pos­sibly only A. stellio, infection failed to established itself in geckoes (P. hasselquistii) fed on infected ticks. The vector tick's identity remains inconclusive. The ticks found on the lizards, by all morphological criteria outlined by Hoogstraal (1956) , are indistinguishable from H. aegyptium, which has been found feeding pre­dominantly on land tortoises (Testudo graeca) and vectors Hemolivia mauritanica (Michel, 1973; Landau & Paperna, 1997) . Although Hoogstraal (1956) notes that rarely the tick was found feeding on A. stellio, in our experiments (unpublished), larvae, nymphs and

Mémoire 25 Parasite, 2002, 9, 17-27

Table IV. - Experimental infection of starred lizards by feeding on H. kisrae sporocysts from ticks.

P A P E R N A I., K R E M E R - M E C A B E L L T . & F I N K E L M A N S.

adults reared from the ticks recovered from the lizards, refused to attach to tortoises. Ball et al. ( 1969 ) found sporozoite stages in the tick Amblyoma dissimile as well as in mosquitoes (Culex tarsalis, Aedes togoito), when fed on same Hepatozoon

fusifex-'mkcted Boa constrictor. The parasites, which developed in the tick, as authors also admit, however, do not necessarily have to be conspecific with the ones found in the mosquitoes. These mosquitoes failed to acquire infection when fed on snakes infected via A. dissimile. Apparently, all true members of the genus Hepatozoon, as well as species o f Hemolivia have a dichotomous course o f merogony, one of active large-progeny mero-gonies to sustain the subsequent gamogonous gene­ration in the blood and the other, yielding persistant cyst-l ike stages. T h e s e cys t -enc losed stages were termed cystozoites (Landau et al., 1972) . They were transmitted when ingested as prey by a subsequent ver­tebrate host (Landau et al, 1972; Smith & Desser, 1998). In our experiments, active merogonic stages could not be separated from the cyst-like stages in the viscera used to infect lizards via feeding. In H. kisrae, active merogonies developed in endothe­lial cells in the lungs and to a lesser extent in the liver. Cystozoites occur only in the liver, mostly in macro­phages in MMC. In A. stellio experimentally infected by Hemolivia mariae, a few cystozoites also developed in the lungs, in formed MMC (Smallridge & Paperna, unpublished). The large-progeny merogonies are formed by polyen-dodyogenous merogony, and generations o f 4 to 16 large meronts are followed by one or more eight to 32 progeny-generations of micromeronts, which escape to the blood to develop into gametocytes. The dizoic cystozoites have been seen to form by endodyogeny (Landau et al, 1972) , as also seen in this study: cystic stage-four and eight zoites are generated by succes­sive endoclyogenies. Deve lopmen t o f the present ly descr ibed spec ies , although taking place in ticks, occurs similarly to that seen in moquitoes, i.e. within the oocyst located in the tissue lining the haemocoel (Bashtar etal, 1984; Lowi-chik etal, 1993; Smith & Desser, 1997) . In Hemolivia developing in ticks the sporocysts released (as sporo-kinetes) from the oocyst remain located inside parasi-tophorous vacuoles within the tick gut cells (Smallridge & Paperna, 2000b) . In ultrastnicturally examined H. kisrae sporocysts, the crystalloid material initially assembled within pockets o f E R , by the same route as seen in species develo­ping in mosquitoes (H. domerguei, Vivier etal, 1972; H. aegyptii, Bashtar et al, 1984) , and similarly led to the formation o f large crystalloid arrays which are split b e t w e e n the ult imately formed sporozoi tes . Although the developmental process in Hemolivia also

involves formation and split o f crystalloid substance between the offspring, it seems to proceed differently. The extensive E R , which accompanies the forming crystalloid bodies in Hepatozoon, is lacking in Hemo­livia (or disappears at an earlier stage o f differentia­tion). In Hemolivia the crystalloid substance is first split between sporokinetes (Smallridge & Paperna, 2000a) , and only then between the sporozoites (Paperna & Smallridge, 2000b) .

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Regu le 27 septembre 2001 Accepte le 12 novembre 2001

Parasite, 2002, 9, 17-27 Mémoire 27